Although the introduction of highly active antiretroviral therapy (HAART) targeting the human immunodeficiency virus (HIV) revolutionized acquired immunodeficiency disease (AIDS) therapy, nucleoside reverse transcriptase inhibitors (NRTIs) that are a cornerstone of HAART cocktails have been found to cause severe mitochondrial toxicities that have limited their use. NRTIs cause toxicity by inhibiting mitochondrial DNA (mtDNA) synthesis through inhibition of polymerase g (Pol g), which is responsible for mtDNA synthesis. Although anti-HIV NRTIs have drawn the most attention to nucleoside drug-induced mitochondrial toxicity, this toxicity has also been seen with other antiviral nucleoside drugs such as fialuridine (FIAU) and clevudine (L-FMAU) that were being developed to treat hepatitis B (HBV) infections, and gemcitabine, the drug of choice for the treatment of pancreatic cancer. Moreover, the problem is likely to become more widespread as nucleoside drugs are used outside the antiviral and anticancer fields to disease areas such as multiple sclerosis. Unfortunately, there is no current proven general treatment of prevention strategy for nucleoside drug-induced mitochondrial toxicity. Thus, the development of innovative approaches to preempt or treat mitochondrial toxicity will be an important advance in the field of nucleoside drug therapy. NRTI triphosphates, the metabolites that inhibit Pol g can be synthesized inside mitochondria from imported NRTIs from the cytosol. Imported nucleosides are sequentially phosphorylated starting with rate-limiting monophosphorylation by mitochondrial-specific thymidine kinase 2 (TK-2) and/or deoxyguanosine kinase (dGK). Mitochondrial membrane nucleoside transporters (NTs) import NRTIs from the cytoplasm. The NT isoforms that have been implicated in mitochondrial nucleoside uptake are the equilibrative nucleoside transporters (ENTs), particularly ENT1 and ENT3. The transgenic overexpression of ENTs has been shown to enhance the mitochondrial toxicity of the former anti-hepatitis B nucleoside drug fialuridine (FIAU) and the NRTI AIDS drug AZT. We have obtained preliminary data showing that a prodrug approach could be used to protect against NRTI-induced mitochondrial toxicity; and we will test that hypothesis by pursuing the following specific aims: 1) synthesize and characterize novel prodrugs for lack of NT inhibition and cellular release of active drug, 2) evaluate the abiliy of prodrugs to protect mitochondria from the toxicity of nucleoside analog drugs, and 3) study biopharmaceutic and pharmacokinetic properties of selected prodrugs and their influence on the pharmacokinetics of nucleoside drugs of interest. A multidisciplinary approach integrating synthetic medicinal chemistry, biochemical and molecular biological assays, laser scanning confocal microscopy, chromatography and tandem mass spectrometry will be employed. The success of the project will confirm the possibility of protecting against nucleoside analog mitochondrial toxicity through the inhibition of nucleoside transporters, as an innovative general preemptive means of protection against toxicities of anti-HIV NRTIs and other nucleoside drugs like the anticancer nucleoside analog gemcitabine, which is associated with rare but potentially fatal liver toxicity and myopathy. Novel research tools will also be developed.